Turning adversity into opportunity: Small extracellular vesicles as nanocarriers for tumor‐associated macrophages re‐education

Abstract Currently, small extracellular vesicles (sEV) as a nanoscale drug delivery system, are undergoing biotechnological scaling and clinical validation. Nonetheless, preclinical pharmacokinetic studies revealed that sEV are predominantly uptaken by macrophages. Although this “sEV‐macrophage” propensity represents a disadvantage in terms of sEV targeting and their bioavailability as nanocarriers, it also represents a strategic advantage for those therapies that involve macrophages. Such is the case of tumor‐associated macrophages (TAMs), which can reprogram/repolarize their predominantly immunosuppressive and tumor‐supportive phenotype toward an immunostimulatory and anti‐tumor phenotype using sEV as nanocarriers of TAMs reprogramming molecules. In this design, sEV represents an advantageous delivery system, providing precision to the therapy by simultaneously matching their tropism to the therapeutic cell target. Here, we review the current knowledge of the role of TAMs in the tumoral microenvironment and the effect generated by the reprogramming of these phagocytic cells fate using sEV. Finally, we discuss how these vesicles can be engineered by different bioengineering techniques to improve their therapeutic cargo loading and preferential uptake by TAMs.


| INTRODUCTION
Tumor-associated macrophages (TAMs) are an important component of the leukocyte infiltrate of tumors that play a multi-functional role in cancer progression, exerting dramatic impacts on tumor initiation and promotion, metastasis, immune regulation, and angiogenesis. 1,2 Derived from circulating inflammatory monocytes and tissue-resident macrophages that are recruited to tumor tissue and then induced to acquire pro-tumoral phenotypes, TAMs are characterized by an M2-like phenotype. 3 Through the secretion of an array of growth factors, cytokines, chemokines, hormones, matrix-remodeling proteases, metabolites, and small extracellular vesicles (sEV), 4 TAMs are not only involved in cancer progression and metastasis but also in the tumor recovery after cytoreductive therapies such as chemotherapy, radiotherapy, and biological therapies. 5 As Cassetta and Pollard discuss in their review manuscript, 5 macrophage infiltration also interferes with the immunotherapy efficacy, neutralizing efforts to reactivate CD8+ T cells in some tumors. In clinical studies, an increase in macrophage density in tumors correlates with markers of poor prognosis and reduced overall patient survival, with some exceptions such as colorectal carcinoma. 6,7 Although several therapeutic strategies have been developed to target macrophages in the tumor microenvironment (TME) at the level of recruitment, survival, or reprogramming toward anti-tumoral activities, 8 they are still under preclinical and clinical evaluation.
Reprogramming TAMs toward an M1-like anti-tumoral phenotype provides the opportunity to balance the leukocyte infiltrate of the tumor stroma, blunting it from a predominantly pro-tumoral state to one with anti-tumoral activity, without the drawback of long-term toxicity caused by the depletion of all macrophages. 5,6 With promising preclinical results, molecules such as PI3Kγ and histone deacetylases (HDAC) inhibitors have achieved the reprogramming of TAMs toward an anti-tumor phenotype state, resulting in a decrease in tumor growth together with increased tumor sensitization to immune checkpoint inhibitors, 9 data that demonstrate the potential clinical efficacy of this therapeutic strategy. 5,6 However, the main barrier to the clinical translation of TAMs reprogramming strategies is the inadequate drug delivery efficiency, consequence of the aberrant vascular architecture, high interstitial fluid pressure, and the compact structure of the extracellular matrix (ECM) in the tumor. 10 Nevertheless, these in vivo delivery barriers could be resolved using specialized drugdelivery methods, 11,12 such as nanoencapsulation, since it is known that nanoparticles or nanovesicles of 100 nm in size are selectively retained in tumor tissue through the mechanism called enhanced permeation and retention (EPR) effect. 13,14 sEV are considered effective nanocarriers of biological origin, both endogenous molecules and those artificially loaded. 15 Characteristics such as their rapid internalization into acceptor cells, lack of immunogenicity even at repeated doses, and the potential for modifying their molecular cargo or their surface membrane for targeting a specific cell or tissue, have turned sEV into powerful therapeutic agents. 16 However, the data derived from the pharmacokinetics studies have reported that exogenous or therapeutic exosomes injected intravenously in rodents accumulate primarily in the liver, spleen, lungs, and gastrointestinal tract, uptake attributed mainly to macrophages resident in these organs. 17 This natural "tropism" of sEV toward monocytes/macrophages represents a barrier to therapeutic strategies that use sEV as nanocarriers of natural or artificially loaded therapeutic molecular cargo by decreasing their bioavailability in the circulation to just a few minutes. [18][19][20][21] Nonetheless, this drawback can be turned into a strategic advantage to TAMs re-education in solid tumors, mainly in those with a high infiltrate of myeloid cells. Taking advantage of these native tropisms that results in a rapid and preferential distribution to macrophages, sEV in their native or engineered state can be used as agents to reeducate these phagocytic cells toward a predominantly pro-inflammatory M1-like phenotype to regulate tumor progression or favor the response of other therapeutic interventions. 8 This sEV-based therapeutic approach has been successfully tested at the preclinical level in a colon carcinoma model. By functionalizing the sEV surface membrane with an antisense oligonucleotide (ASO) targeting the transcription factor STAT6, the reeducation of TAMs from their native M2-like phenotype to an M1-like phenotype with potent antitumor properties is achieved. 22 In this review, we summarize the role of TAMs in the TME and the clinical relevance of these cells as a potential therapeutic target.
Specifically, we discuss how the tumor-supportive phenotype of TAMs can be reprogrammed using engineered sEV as a drug delivery nanocarrier. For this purpose, we present different experimental techniques that modify the cargo and surface of sEV and candidate therapeutic molecules with a reported ability to reprogram TAMs in a wide variety of cancer models.

| PLASTICITY AND DIVERSITY, THE CURRENT MACROPHAGE PERSPECTIVE
Macrophages are myeloid cells from the innate immune system that have an essential role in tissue homeostasis, regeneration, inflammatory response, and elimination of pathogens, among others. Originally, it was thought that macrophages shared their origin with macrophage dendritic cell precursors and bone marrow (BM)-derived peripheral monocytes; however, during the last years, murine models have demonstrated that different tissue-resident macrophages populations develop from hematopoietic progenitors present in the yolk sac independently from the BM. 23,24 Macrophages are highly plastic cells that can integrate multiple signaling pathways simultaneously depending on the stimuli, altering their transcriptional networks and function through epigenetic modifications. 25 Complementarily, today it is well established that macrophages can reprogram their transcriptional profile by altering only their microenvironment stimuli, results that have been validated both in vitro and in vivo. 26 The array of functional phenotypes by which these cells respond to the environment changes is known as the macrophage polarization process . 27 Since in preclinical and clinical studies the coexistence of different, unique, and even mixed polarization states had been reported a dynamic and reversible mechanism is suggested to regulate the macrophage polarization. 28 Thanks to the advancement of single-cell techniques, new evidence supports the presence of a wide diversity of macrophage subpopulations within the same tissue, where different subpopulations regulate physiological and pathological processes. 29 For these reasons, it is increasingly challenging to attribute a function to a certain phenotype, highlighting the need for the nomenclature in the study of these myeloid cells.
In the early 2000s, the M1/M2 concepts were introduced based on the ability to produce NO in the presence of LPS in vivo. 30 Nowadays, this binary nomenclature is applied in more generalized ambits, where different populations of macrophages are cataloged with these terms. Even though this overgeneralized classification misses the macrophage diversity, it is still one of the most common nomenclatures in the literature. Another nomenclature in macrophages is based on the activation stimuli and the expression of certain markers. 31 In this sense, the classical activation pathway corresponds to the incubation of macrophages with IFN-γ and LPS in vitro, inducing the differentiation to M1-like macrophages with increased production of NO through the expression of NOS2, increased antibody-dependent cellular phagocytosis (ADCP), membrane concentration of MHCII and secretion of proinflammatory cytokines (IL-1, TNF, IFN), among others. 31 Instead, the alternative activation pathway that originates the M2-like macrophages corresponds to the incubation with IL-4 in vitro, increasing the expression of Arg1 (inhibitor by competition of NOS), the secretion of anti-inflammatory cytokines (IL-4, IL-10, IL-13, among others), and immunosuppressive and angiogenic factors. 31

| ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN HUMAN CANCER
The TME is a complex ecosystem in which tumor cells coexist with a heterogeneous cell population composed of multiple cell types like endothelial cells, stromal cells, and immune cells. 32 Macrophages, polymorphonuclear cells, mast cells, natural killer, dendritic cells, and T and B lymphocytes are the immune cell components that essentially determine the tumor's fate and the progression of the metastatic disease. 33 In fact, the immune cell composition and organization within the TME are correlated to clinical outcomes in cancer patients. 33 TAMs and their precursors are the most abundant population of the tumor-infiltrating immune cell in many solid tumors, as shown by immunohistochemical analyses of the TAMs marker CD68 + and by CIBERSORT-mediated dissection of gene expression profile. 5,34,35 TAMs are recruited into the tumor by chemokines secreted by cancer and stromal cells, playing an essential role in the regulation of cancerrelated inflammation. 36 Different subpopulations of TAMs act as a source of local and systemic cues to support the tumor angiogenesis, proliferation, survival, and invasiveness of tumor cells; and suppression of cytolytic T-cell responses. 35 This assortment of pro-tumor functions is consistent with the results of clinical studies showing that human macrophage density in tumor samples is associated with poor prognosis. 37 In the last 5 years, the correlation between TAM infiltration and progression in cancer has been confirmed by several meta-analyses performed in breast cancer, 38 cervical carcinoma, 39 gastric cancer, 40 Hodgkin lymphoma, 41 pancreatic cancer, 42 and lung cancer 43 but not in colorectal cancer. 44 Like macrophages in normal tissues, TAMs are characterized by their great phenotypic and functional plasticity that allows them to adopt a pro-inflammatory M1-like state, or an anti-inflammatory M2-like state or even an intermediate activation state depending on the environmental conditions to which they are exposed locally in the TME or those that occur during a treatment. 5,34,45 Furthermore, a recent analysis of the tumor-immune signature shows that different subpopulations of TAMs that coexist in the TME often co-express canonical pro-inflammatory M1 and alternatively activated M2 genes in individual cells. 34,[46][47][48] Although the M2-like pro-tumoral TAMs is the phenotype predominantly found in tumors, in the very early phases of oncogenesis the foremost polarization of the TAMs is toward the M1-like state, which mediates multipronged anticancer effects. 34,36,49 However, this permanent state of pro-inflammatory activity induces genomic instability in cancer cells, which in consequence acquire the ability to repolarize TAMs toward an M2-like state. 50 This repolarization of TAMs favors tumor progression and malignancy through the secretion of growth factors, cytokines, and chemokines-such as transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), M-CSF, IL-10, and chemokine C-X-C motif ligand (CXCL) 51 -hormones, matrix-remodeling proteases, metabolites, and sEV. 4,37 A distinct subpopulation of TAMs with different transcriptome and cell surface markers stimulates different aspects of carcinogenesis such as angiogenesis, immune evasion, invasion, and metastasis 2,6,37,52,53 ( Figure 1). However, its presence in TME also regulates the response to treatments. In vivo studies showed that the pro-tumoral phenotype of TAMs mediates acquired resistance of cancer cells to chemotherapy and radiotherapy providing them with survival factors that activate antiapoptotic programs in malignant cells. 51 In fact, selective depletion or inhibition of TAMs results in a reduction of the resistance associated with chemo and radiotherapy. 51 Furthermore, TAMs have also been considered regulators of immunotherapy since the immunosuppression they cause acts as a barrier to tumor sensitivity to cancer immunotherapies, thus inducing acquired resistance and immune evasion of the tumor. Although the mechanisms contributing to the immunotherapy resistance are still under investigation, it is known that macrophages express elevated levels of CTLA-4 (an immune checkpoint receptor), which are associated with the downregulation of anti-tumor activities of T-cells. 54 In addition, CD68+ macrophages are the predominant immune cells expressing PD-L1, 55 an immune regulatory molecule that interacts with PD-1 on T cells at the immunological synapse. Activated by TME-derived factors, these PD-L1+ TAMs mediate CD8+ T-cell dysfunction through the PD-1/PD-L1 axis, observations reported in several types of cancer as hepatocellular carcinoma, 56 ovarian cancer, 57 bladder cancer, 58 soft tissue sarcoma, 59 head, and neck squamous cell carcinoma, 60 and cholangiocarcinoma. 61 Also, TAMs express various other checkpoints ligands such as PD-1, 62 PD-L2, 63 B7-S1, 64 galectin-9, 65 and V-domain Ig-containing suppressor of T-cell activation (VISTA) 66 that inhibit the anti-tumor immune response at different levels. 67

| TUMOR-ASSOCIATED MACROPHAGES AS THERAPEUTIC TARGETS
Since TAMs play a crucial role in the regulation of cancer-related inflammation and interfere with the beneficial outcomes of current therapeutic strategies, TAMs represent a target cell population in new anti-tumor therapeutic approaches. Other features like its great abundance in the tumor, its genomic stability, and rapid response to external stimuli reflected in its extreme plasticity highlight its potential as a therapeutic target for cancer. 8 Reprogramming TAMs toward an M1-like anti-tumor phenotype leads to a rebalancing of the composition of TAMs in TME toward a predominantly pro-inflammatory population that limits tumor progression and increases anti-tumor immune responses.
One of the first studies to show that TAM re-education within TME exerts an effect on tumor regression was reported in 2013, using a colony-stimulating factor 1 receptor (CSF-1R) inhibitor-called BLZ945-in multiple preclinical glioblastoma models. 68 After the inhibition of CSF-1R, the authors observed a blockage in the growth and progression of the glioma, however, surprisingly this anti-tumor effect was not mediated by the depletion of TAMs but rather by their re-education within the glioma microenvironment. Unlike macrophage depletion, which was observed in healthy tissues (including the brain), transition that allows controlling tumor growth in vivo. 70 In the same The pro-tumor phenotype of TAMs promotes tumor development through multiple interactions with TME. TAMs correspond to the most abundant infiltrating immune cell population in TME, exerting various actions in favor of carcinogenesis. Through direct and indirect interactions (through the secretion of growth factors, cytokines, and interleukins, among others) with the other infiltrating cell populations in the TME, the pro-tumor phenotype of TAMs contributes to greater survival and proliferation of tumor cells, as well as in the promotion of malignant characteristics such as invasion and metastasis. By located in predominantly hypoxic regions in the TME, TAMs also have strong pro-angiogenic properties that help support tumor development and progression. Also, TAMs create an inflammatory and immune-evading environment, interfering with the response to antitumor treatments, especially immunotherapy. CTSB, cathepsin B; CTSS, cathepsin S; ECM, extracellular matrix; EGF, epidermal growth factor; HGF, hepatocyte growth factor; IL-10, interleukin 10; IL-1β, interleukin 1 beta; IL-6, interleukin 6; IL-8, interleukin 8; MMP2, matrix metalloproteinase 2; MMP7, matrix metalloproteinase 7; PDGF, platelet-derived growth factor; PIGF, placenta growth factor; TGF-β1, transforming growth factor-beta 1; TNF-α, tumor necrosis factor-alpha; VEGF-A, vascular endothelial growth factor A (created with BioRender.com) Recently, Wang et al. 73 also described an interesting TAM reprogramming strategy evaluated at the preclinical level but with an interesting translational approach. Using a nucleic acid carrier system with an affinity for mannose receptors in TAMs/tumor-infiltrating dendritic cells (TIDC) and that specifically responds to the low-pH tumor microenvironment, 74 the authors achieve TAM-targeted delivery of miR-99b and/or miR-125a. After the effective transfer of these miRNAs to TAMs, the growth of an orthotopic tumor of murine hepatocellular carcinoma (HCC) and subcutaneous Lewis's lung cancer (LLC) was significantly prevented by re-education of TAMs toward an anti-tumor phenotype (M1-like state) with enhanced immune surveillance. 73 The studies of the mechanism of action revealed that miR-99b might promote M1 while inhibiting M2 polarization by downregulating the signaling pathways of κB-Ras2 and/or mTOR, respectively. Interestingly, the authors reported that miR-99b might amplify M1 macrophage function through NF-κB by a positive feedback regulation loop, resulting in increased phagocytosis and antigen presentation.
Other immunomodulatory agents such as interleukin-12 (IL-12) 75 or CD40 agonists 76 have also been reported to induce repolarization of TAMs. However, since these immunomodulatory molecules can activate different cell types, they are associated with dose-limiting adverse effects and systemic toxicities. 77 Other strategies used to repolarize macrophages are described in Table 1, which summarizes the main biological findings in a cancer context. Given that in the literature it is possible to find several review articles that describe in detail the different molecules that exert their effect in the re-education of TAMs, we present in Table 1  Among them, sEV are the nanovesicles that are currently revolutionizing the field of biomedicine due to their numerous advantages as carriers of therapeutic molecules. Attributes as their stability, biocompatibility, permeability, low toxicity, and low immunogenicity determine its success as a nanoparticle drug delivery system. 16 sEV are non-self-replicative lipid-based vesicles generated from different subcellular compartments with a demonstrated role in cell-cell communication. 15  For example, it has been described that after 24 h of systemically injected HEK293T DiR-labeled sEV, 3% of the total tissue fluorescence was detected in the tumor in a melanoma murine model. 17 Likewise, unpublished data from our laboratory also shows that DiR-

| ENGINEERED sEV TO SELECTIVELY REEDUCATE TAMS TOWARD ANTI-TUMOR ACTIVITY
Enriching sEV with specific reprogramming molecules to efficiently promote reeducation of TAMs toward an M1-like phenotype is a plausible strategy to achieve TEM reform toward one with proinflammatory activity permissive to the action of the immune system.
Indeed, this strategy should be designed to impact TAMs preferentially or selectively, without affecting circulating monocytes or tissueresident macrophages, to limit the potential side effects due to an off-target interaction. Both loading with specific molecules to achieve the desired therapeutic efficacy and selective targeting to TAM to ensure therapeutic safety are the fundamental pillars on which modifications of sEV should be designed. Fortunately, native sEV can be modified both in their molecular cargo and in their surface, membrane using different biotechnological tools with variable influence on the integrity of the membrane or the bioactivity of the therapeutic payload.

| Enrichment of sEV with TAM reprogramming molecules
As shown in Table 1 pro-inflammatory phenotype. Indeed, the efficacy of these drugs can be potentially improved through a delivery platform using sEV that allows their accumulation in tumor tissue due to the EPR effect. These therapeutic molecules can be enriched into sEV, either by "loading" them inside or onto their surface to achieve controlled and efficient delivery to the tumor niche. Several excellent manuscripts can be found in the literature that review these different methodological techniques in-depth, [110][111][112][113] so here we will only analyze them briefly, presenting the advantages and disadvantages of each of them (Table 3). In general terms, "loading" techniques seek to enrich the cargo of sEV or on their surface with specific endogenous or exogenous molecules of natural or artificial origin (as small molecules, drugs, proteins, or nucleic acids) to increase their effective transfer to the acceptor cell or target tissue to trigger a desired biological effect. These techniques can be classified into two major approaches: pre-and postisolation techniques. In the pre-isolation techniques or cell-based loading methods, the starting material is the parental cells; therefore, the enrichment with a specific cargo occurs before the isolation of the sEV.
On the contrary, in the post-isolation techniques or noncell-based loading methods, sEV are the direct starting material for specific molecular enrichment; thus, modifications occur after isolation of sEV by ultracentrifugation, gradient ultracentrifugation, size-exclusion chromatography, co-precipitation, or field flow fractionation.

| Pre-isolation techniques
Using cell-based loading approaches, sEV can be modified to load therapeutic proteins directly into their cargo or display them on its surface. 16 To carry out these modifications, methods that use molecule sorting modules (MSMs) for sorting specific proteins and RNAs to sEV, or a nonspecific sEV enrichment methodology without involving modules for packaging and sorting can be used. Regarding the latter, the genetic editing of sEV-producing cells is usually used to enrich specific proteins or nucleic acids, a strategy that involves the transfection of the parental cell with a gene of interest. 112 Likewise, to load specific drugs, direct co-incubation of the parental cell with the drug allows its encapsulation in the sEV and subsequent secretion. 114 Regarding the methods that use MSMs, to guide a protein to the surface of sEV, a signal peptide is commonly used. For example, lysosome-associated membrane protein 2b (Lamp2b) is a sEV surface protein with a signal peptide that has been widely used to display any Although less complex than pre-isolation approaches, these techniques certainly require experience working at the nanoscale. As direct loading methods, incubation, sonication, extrusion, electroporation, freeze-thaw treatment, and dialysis are strategies with variable efficiency to load exogenous molecules into sEV (efficiency that depends on the intrinsic properties of the cargo such as hydrophilicity, hydrophobicity, and molecular weight) and with variable alteration of the integrity and functionality of the sEV, attributes that must be verified after making modifications.
Incubation is the most straightforward approach to loading a specific cargo on sEV by diffusion through a concentration gradient. Using particular times, temperatures, and pH, different laboratories have successfully loaded hydrophilic and hydrophobic molecules. 113 Since sEV contains a hydrophilic core and lipid bilayer in its membrane, hydrophilic drugs tend to incorporate into the aqueous phase within sEV. In contrast, hydrophobic drugs are more stable in the lipid-enriched membrane. Some molecules that have been successfully loaded into sEV are doxorubicin, 121 paclitaxel, 122 curcumin, 123 withaferin A, 123 anthocyanidins, 123 nucleic acids, 121,124 proteins, and peptides. 125 Among the physical procedures described to load the sEV are electroporation, sonication, surfactant treatment, freeze-thaw treatment, extrusion, and dialysis. Electroporation, sonication, and surfactant treatment generate pores in the phospholipid bilayer of sEV, which allows the incorporation of exogenous cargo. The freeze-thaw treatment, extrusion, and dialysis allow enriching the cargo during the membrane recombination processes. These methods have a variable loading efficiency that must be verified and ideally quantified, with moderate alterations in the integrity of the membrane that must be corroborated before starting any preclinical study.
Electroporation is a physical technique that uses an electric field in the sEV resuspended in a conductive solution to generate temporary micropores, allowing the membrane's permeability and the diffusion of the exogenous cargo into the sEV. Using this technique, the sEV have been successfully loaded with drugs as doxorubicin, 126 5-fluorouracil, 127 and paclitaxel 128 ; nucleic acids 17,22,104,105,[107][108][109]127,129,130 ; proteins 131 ; and theranostic nanoparticles for cancer. 132 Sonication is a procedure that applies a mechanical shear force to weaken the integrity of the sEV membrane, which promotes the diffusion of exogenous molecules into the cargo of the nanovesicles. Such is the case of small molecules such as gemcitabine, 133 doxorubicin, 134 and paclitaxel 128,134,135 ; proteins as catalase 136 ; and various nanomaterials as gold nanoparticles. 136 Surfactant treatment using, for example, saponin or triton allows to increase of the permeability of the sEV' membrane through the formation of complexes between the surfactant molecules and the cholesterol of the membrane.
Using this procedure, proteins have been successfully loaded. 131,136 The freeze-thaw strategy is a simple way to encapsulate into sEV various types of molecules such as drugs, proteins, and peptides. 136 Through repetitive freeze-thaw cycles, it is also possible to fuse the liposome membranes (which carry the modification of interest) with the sEV membrane to develop a hybrid sEV. 137 Extrusion is a process that mixes the sEV with the exogenous cargo in a lipid extruder, physically inducing the rupture of the sEV membrane and its subsequent mixing with the exogenous cargo as proteins. 131,136 Finally, dialysis allows to enrich the cargo of sEV with a drug of interest by mixing them on dialysis membranes or tubes, and then dialyzing them with stirring to obtain drug-loaded sEV. 113 Using this methodology, nucleic acid, specifically miRNA, siRNA, and single-stranded DNA (ssDNA) have been loaded. 138

| Selective targeting of TAMS using engineered sEV
In the same line, the surface membrane of sEV can be altered by direct conjugation or genetic modifications in the parental cell. For this, click chemistry or ligases have been used to directly attach molecules to the sEV surface through covalent bonds to provide them a F I G U R E 2 Engineering sEV for targeted drug delivery. Small extracellular vesicles are a versatile platform for drug encapsulation, possessing multiple advantages over conventional synthetic nanocarriers for drug delivery. As an anti-tumor treatment, TAMs reprogramming molecules can be "loaded" inside or on the surface of the sEV to achieve their controlled and efficient delivery to the tumor niche. These loaded sEV will act as re-educators of TAMs from their pro-tumoral phenotype (M2-like) toward an anti-tumor phenotype (M1-like). There are various techniques to enrich or load exogenous molecules to sEV, classified into two main types: pre-isolation and post-isolation techniques. In the former, the modifications are carried out at the level of the parental cell, which secretes sEV with the modifications designed. While in the second, the modifications are made directly on the sEV, after their isolation and purification (created with BioRender.com) desired functionality. 16,139 This method has allowed to attach peptides to the phospholipid membrane to target specific anatomical sites. 140,141 Targeting M2-like receptors-like CD206-in TAMs is the current most common strategy studied to increase therapeutic nanoparticle delivery. Using peptides like "UNO" 71  Another tumor-specific property is the over-expression of certain proteases like urokinase-type plasminogen activator (uPA), membranetype serine protease 1 (MT-SP1/matriptase), and legumain, which had been reported in several human tumors. 144 To avoid the adverse effects associated with CAR-T therapies, Han et al. 145 designed a masked chimeric antigen receptor with a linker suitable to be cleaved by these proteases, resulting in a fourfold increase in the binding to its objective antigen after a protease treatment in vitro, decreased lung tumor growth in vivo and overall increased survival rate compared to its controls. In the context of a TAM's re-education therapy using sEV, a similar strategy applying the cleavable linker could be evaluated.
Recently, the Prostaglandin F2 Receptor Negative Regulator (PTGFRN) was identified as a "scaffold" protein suitable for genetically engineering sEV with defined therapeutic properties. 146 Also, self-peptides had been attached to the sEV surface by direct conjugation that act as a "don't eat me" signal, improving systemic circulation rate and decreasing the internalization by phagocytic cells. 139

| PERSPECTIVES AND FINAL CONCLUSIONS
The use of sEV as a nanoparticle drug delivery system is in full swing of study, development, and validation in biomedicine. As a nanocarrier, its success is based on its attributes such as its stability, biocompatibility, permeability, low toxicity, and low immunogenicity. However, biodistribution and pharmacokinetic studies of sEV show that they have a short half-life in the systemic circulation, accumulating mainly in organs associated with the mononuclear phagocyte system (liver, spleen, lung).
Macrophages are primarily responsible for their clearance, being considered a barrier to the bioavailability of sEV and, consequently, the therapeutic molecules they transport. This natural "tropism" that exists between sEV-macrophages, although it may represent a disadvantage in its bioavailability as a carrier, it also means a strategic advantage for those therapies that involve macrophages. Indeed, TAMs are not immune to this (favorable) interaction with sEV, which has shown that in the TME, these phagocytic cells have a preferential uptake. Undoubtedly, to minimize the potential side effects associated with off-target interaction is essential that sEV-based therapies should consider specific targeting on M2-like TAMs without significantly affecting M2-like macrophages resident in healthy tissues (located, for example, in the liver, spleen, or lung). Given the similarities between both cell populations, the nanovesicles drug delivery system should incorporate in their design modifications that allow increasing the control of activity in the TME, taking advantage of tumor-specific properties such as acid pH or expression of specific proteases. Only in this way could one have control of the therapeutic precision.
Parallel to sEV design and engineering developments, it is imperative to advance in the productive scaling up and industrialization of clinical-grade sEV production. The purpose is to expand and ensure the availability of therapy to all patients at the right time, either in monotherapy or in combination with other existing or emerging therapies. For this, it is essential to optimize the different stages of the production process to achieve greater efficiency and production performance of sEV, with high purity and maintaining its identity.
Thus, for example, in upstream processing, the choice of

CONFLICT OF INTEREST
Francisca Alcayaga-Miranda received stipends from Cells for Cells.
The other authors have no conflicts of interest to declare.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
This article does not contain any studies with human or animal subjects.